Note: Descriptions are shown in the official language in which they were submitted.
1~3464~
FIELD OF THE INVENTION
; This invention relates to pheromone monitoring
systems, and more particularly to methods of detecting and
monitoring the quantity of pheromone substances in a gaseous
or liquid environmental sample.
BACKGROUND OF THE INVENTION
It is known that animals secrete substances known as
pheromones, to influence the behaviour of other animals of the
same species. Insects, for example, secrete sex pheromones, in
extremely low levels, to attract mates of the opposite sex.
The mechanism of production and secretion of sex pheromones
by insects is still incompletely understood. They appear to
be secreted by moths or beetles during mating periods, at a
particular daily calling stage. Insect population control
systems have recently been proposed, based on the use of the
sex pheromones of the insects concerned.
Some insects that cause damage to forestry and
farming products have been reported to secrete long chain
unsaturated aldehydes that function as sex pheromones, either
alone or in combination with other substances. Examples
include such major pests as the corn earworm (cotton bollworm),
the dermestia beetles, the spruoe budw~rm and the tobacco budworm. Consequently,
methods have been developed to monitor or control the population
of these pests by use of the appropriate sex pheromone
substance, either synthetically or naturally produced. Lures
~a~
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have been made, containing the sex pheromone, optionally in
admixture with a solid or liquid carrier, in which the insects
are trapped and counted so as to monitor the population.
Infested areas may be sprayed with sufficient sex pheromone
to confuse the insects and prevent their mating and reproduction.
The sex pheromone may be included in a pheromone trap, in which
insects are collected for killing, e.g. by inclusion of
insecticide in the trap.
Further developments and studies of sex pheromones
and their potential uses in insect population control are,
however, hampered by the lack of a suitable method for deter-
mining the concentrations of the pheromones being used.
Effective concentrations of pheromones for this purpose are
extremely small, in many cases below the minimal range. Before
the efficiency and potential utility of any method of insect
population control based upon the use of sex pheromones can be
properly assessed and developed, there needs to be a method
which can be used in association with the pheromone which will
permit the accurate and rapid detection and monitoring of the
pheromone substances. Only in this way can efficiency of traps
be established, and the necessary effective concentrations of
pheromones be established and hence applied.
BRIEF DESCRIPTION OF THE PRIO~ ART
The current practice for monitoring pheromone con-
centrations is by means of gas-liquid chromatcgraphy. In
such processes, a sample of atmosphere is collected by
642
absorption from the air, extracted with an organic solvent, in
some instances derivatized to a form more easily detected on
analysis, and then subjected to gas chromatography. Such a
procedure is difficult and time-consuming, requiring the
conducting of several steps of sample preparation. Furthermore,
such methods are generally not sensitive enough to detect the very lcw
concentrations of sex pheromones effective in the atmosphere as
insect attractants.
It is kncwn that long chain aldehydes will react with reduced
flavine mononucleotide, in the presence of oxygen and a
bacterial luciferase enzyme, to oxidise the flavine mono-
nucleotide substrate and the aldehyde, with the emission of
light, at 490 nm. This phenomenom is generally known as
bacterial bioluminescence. Descriptions of this process are
to be found in the academic literature, for example in a paper
by J. W. Hastings, "Annual Review of Biochemistry", 1968,
Volume 37, Pages 597-630, and in a paper by J. W. Hastings and
K. H. Nealson, "Annual Review of Microbiology", 1977, Volume 31,
Pages 549-595. There have also been reports that certain long
chain fatty acids, specifically myristic acid, will also cause
in vivo bacterial bioluminescence presumably by being converted
"in vivo" to the corresponding aldehyde.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery
that, with suitable modifications, the light emitting reaction
of oxidation of a reduced flavine nucleotide catalysed by a
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luciferase enzyme can be conducted in the presence of a sex
pheromone aldehyde, and can be used to monitor very accurately
the amount of sex pheromone aldehyde present, down to extremely
small quantities. It has been found that the intensity of
emitted light is a function of the quantity of sex pheromonal
aldehyde present. By the process of the invention, quantities
of sex pheromone aldehyde as low as 0.1 picamoles (10 -13moles)
or 20 picagrams can be readily determined. Moreover, by
analysing the decay characteristics of the emitted light after
it reaches its maximum intensity, the identity of the phero-
monal aldehyde may be determined.
Thus according to the present invention, there is
provided a process of analysing a liquid sample for content of
aldehyde of sex pheromonal origin which comprises:
preparing a solution of reduced flavine mononucleotide
and luciferase enzyme;
treating said solution with hydroxylamine;
reacting the so-treated solution with said aldehyde-
containing liquid sample in the presence of water and oxygen,
to cause oxidation of the reduced flavine mononucleotide with
emission of light;
and determing the intensity of said light emission,
to determine therefrom the content of aldehyde in said liquid
sample.
The amount of sex pheromonal aldehyde to be deter-
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mined in the monitoring of pheromonc traps, down to 0.1
picamoles, is so low that special methods of assay and pre-
cautions in connection therewith have to be adopted in order
to ensure that all of the aldehyde detected is derived from
the sex pheromone being monitored. Thus, special precautions
have to be taken to remove background aldehyde contaminations.
In accordance with the invention, hydroxylamine is used for
this aldehyde scavenging process, and special methods and
procedures for reducing the flavine mononucleotide and pre-
paring the reaction solutions for the light-emitting lumines-
cence reaction are used. The method of the invention is then
accurate and sensitive enough, to allow quantitative detection
and measurement of sex pheromonal aldehydes down to the
necessary low concentrations for insect trap monitoring.
Thus in the process of the invention, the reduced
monoflavine nucleotide is prepared in admixture with the
enzyme luciferase. This mixture is scavenged for residual
aldehyde with hydroxylamine. Then the sample of sex pheromonal
aldehyde in water is added. The water contains the required
amount of oxygen for the luminescence reaction. Surprisingly,
it is found that the sex pheromonal aldehyde in the sample
reacts with the reduced monoflavine nucleotide to give light
emission of intensity dependent quantitatively on the amount of
sex pheromonal aldehyde, despite the presence in the reaction
mixture of residual amounts of hydroxylamine, the background
1134~i~Z
aldehyde scavenger. It would have been expected that the
residual hydroxylamine would have reactedextensively with the se~
pheromonal aldehyde, to interfere with the reaction
between the sex pheromonal aldehyde and the reduced flavine
mononucleotide, but surprisingly this does not happen. This
feature permits the necessary thorough scavenging of background
aldehyde with hydroxylamine to give the required accurate
determinations of sex pheromonal aldehyde, but also dictates
certain conditions on the order of addition of reagents in
the process of the invention. Thus, it is necessary to avoid
having the test sample and hydroxylamine present in admixture
in the reaction medium in the absence of the reduced flavine
mononucleotide and bacterial luciferase enzyme. For example,
pre-mixing of the liquid test sample and the enzyme solution,
; followed by addition of the reduced flavine mononucleotide thereto, a
procedure which would normally be adopted to give increased sensitivity,
cannot in fact be adopted sinoe this precludes scavenging of the enzyme
solution with hydroxylamine. Only by the procedure of the
invention can the scavenging with hydroxylamine of all the
reagent solutions, necessary to give results of sufficient
accuracy, be properly conducted.
DESCRIPTION OF THE PREFERRED EMsoDIMENTs
Whilst there are several known and acceptable methods
for making reduced flavine mononucleotide, the solution of
; reduced flavine mononucleotide is preferably prepared by
reduction of the flavine mononucleotide, in the presence of
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the luciferase enzyme, with sodium dithionite. This is best
conducted shortly before the light-emitting reaction with the
aldehyde test sample to be conducted. Sodium dithionite, a
solid, powdery material under normal conditions, does not have
an adverse effect on the luciferase enzyme, at least over the
short term. It is preferred to include enzyme stabilizers
such as mercaptoethanol, and phosphate buffers to maintain a
pH of 6-8, in the solution, to ensure stability of the enzyme
therein. Since the assay method of the invention may require
to be conducted under field conditions, it is desirable to
use convenient solid powdered reagents, such as sodium
dithionite where available, and as many premixed solutions
as possible. Other methods for reduction of flavine mono-
nucleotide, such as bubbling gaseouS hydrogen through a
solution thereof, are known and can be used under certain
circumstances. ~lowever, bubbling hydrogen will deactivate the luciferase
enzyme, and so this method cannot be used with a premixed
flavine mononucleotide-luciferase solution. lt is also
satisfactory to form a premixed solution of the luciferase
enzyme and appropriate buffers with the hydroxylamine, and then
mix this solution with flavine mononucleotide before or after
it has been reduced.
This reaction is suitably conducted at or about
room temperature or field temperatures, provided that the reagents
and solutions are kept in the liquid phase. Slightly elevated
1~34164Z
temperatures may offer certain advantages. The reaction should
be conducted at pH at which the enzyme is stable, normally
approximately neutral.
After mixing of all of the necessary reagents and
sample, light is emitted as a result of the flavine oxidation
reaction. The emitted light rapidly rises to a maximum
intensity, and then decays in intensity. The aldehyde con-
centration in the sample is determinable from the maximum
light intensity. By analysis of various features of the
light intensity-decay curve, in the absence of interfering
impurities, characterization of the aldehyde can be obtained,
by comparison with standard curves.
The specific choice of species of luciferase enzyme
is made in accordance with the nature of the pheromonal
aldehyde to be detected. The light emitting response of the
reaction mixture is different, depending upon the chosen
luciferase enzyme. It appears that each luciferase enzyme
species responds over a relatively narrow range of chain
length aldehydes, one luciferase enzyme being preferred over
the range C10-C16 chain aldehydes, and another through the
range C14-C20.
The bacteria luciferase enzymes which are used in
the process of the invention comprise a relatively small, but
kncwn and characterised group. They are obtained and purified from marine
bacteria, being synthesized as the bacteria grow. Samples of
113~64Z
suitable marine bacterla are available from standard culture
collections. The enzymes may be stored for extended periods
of time, under cool conditions, in buffered solutions e.g.
aqueous glycerol solutions. They may be prepared for use
by dilution into aqueous solution, in the absence of glycerol,
suitably buffered. The term "bacterial luciferase enzyme"
refers to an enzyme of bacterial origin/ which will catalyze
the reaction of reduced flavine mononucleotide and aldehyde in
the presence of oxygen with the emission of light. At least
six of these enzymes have been purified and characterized.
The best ones for use in the process of the present invention
are those giving high sensitivity, with a low response in the
absence of exogeneous aldehyde and a high response in the
presence thereof. Routine screening tests of the available
enzymes will readily show those best for use in the inven-
tion under any specific conditions.
The sex pheromonal aldehydes which have been identi-
fied for four major insect pests/ namely the dermestia beetles,
; the corn earworm,the spruce budworm and the tobacco budworm,
to which this invention is especially applicable/ all have
chain lengths of 14 to 16 carbon atoms, and internal un-
saturation. The aldehyde's unsaturation should not be
closer to the chain end than the 5-position/ for reaction
with the luciferase enzyme in the assay system of the present
invention.
1~34~ Z
In another embodiment of the invention, the sex
pheromonal substance to be detected is initially secreted by
the insect in a form other than aldehyde. It may, for example,
be secreted in the form of a long chain alcohol, acid or ester.
Indeed, the biological precursor of the sex pheromonal aldehyde
is in many cases believed to be the corresponding alcohol.
The major component of the sex pheromone of the silkworm is
a long chain alcohol. In such cases, there is included in
the process of the present invention a step of converting the
pheromone into an aldehyde ready for assay with reduced
flavine mononucleotide and luciferase enzyme. The preferred
method of such conversion is by means of an enzyme, e.g.
horse liver alcohol dehydrogenase, when the initial substance
is in alcohol form, or a suitable chemical reagent.
In addition to detection of pheromonal aldehyde in
atmosphere, by collection thereof in a liquid form, the
process according to the present invention is also useful in
the determination of pheromonal contents of insect glands, for
study of the pheromonal emission cycles of the lnsect. The
measurement of pheromone levels in insects is one of the first
steps in the study of pheromone biosynthesis and release. Such
studies are important for the effective application of
pheromones for the control of insect populations and the
prevention of damage to forests and farming crops. The assay
according to the present invention is sensitive enough to
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1~3464Z
measure quantitatively the pheromone levels in the gland of a
single insect such as spruce budworm. The pheromone may be
obtained from the spruce budworm by obtaining a heptane or
hexane extract of the gland, in which the major component of
the spruce budworm pheromone (E~ tetradecenal) is soluble,
evaporati~g off the organic solvent, re-dissolving the residue
in water, and subjecting this solution to the assay method
of the invention. The amount of pheromone obtained from a
heptane extract of the gland of the single spruce budworm is
in the range of 0.4 to 7 0 pmoles. Heptane extracts of the body
of the same insect contain little if any pheromone ~0-0.4 pmoles)
The chemical reactions which occur on subjection of
reduced flavine mononucleotide to aldehyde, oxygen and
luciferase enzyme are incompletely understood, but are
believed to involve the initial reaction of the mononucleotide
with molecular oxygen to form an unstable oxidized interme-
diate, followed by reaction with the aldehyde and molecular
rearrangement to form a hydroxyl-substituted mononucleotide
and a fatty acid. Light is emitted spontaneously at the
molecular rearrangement stage, it is believed.
- The methods of collection of sampies for use in the
present invention are generally as used in the prior art, with
other monitoring methods. Cold traps for condensation of atmos-
pheric samples therein can be used, but may be difficult to
operate under field conditions. It is preferred to absorb
the aldehyde from the
il346 ~Z
atmospheric sample onto a suitable absorbent, for example by
drawing the atmospheric sample, of known volume, through a
gas chromatography column, packed with Porapak Q, then
extracting the absorbent with a suitable organic solvent
such as hexane or heptane, subsequently evaporating off the
solvent and dissolving the residue in water.
The detection and analysis of the emitted light
can be conducted using any known suitable light detection
system for luminescence studies (i.e. a photo-multiplier
tube). When quantitative determinations of aldehydes
are the only required measurement, it may be equipped
with a digital readout of maximum light intensity.
When it is desired to study other parameters, the detector
may be coupled to an automatic plotter to print out a full
light intensity emission and decay curve. From this curve,
the aldehyde amount can be determined from the intensity
maximum. The decay rate and curve should give information
concerning the identity of the aldehyde present. Thus with
an aldehyde sample of unknown type and amount, by use of
various luciferase enzymes and comparison of curve shapes
with standard curves, the type and amount of aldehyde can
often be elucidated. The decay rate, e.g. from 80% to 40%
of intensity maximum, is especially useful in this regard.
The accompanying figures are graphical representa-
tions of decay curves and the like, derived from specific
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samples as detailed below in the specific examples, given by
way of illustration only.
EXAMPLES
Samples for test were generally obtained by the
following procedure.
Air was passed over two micrograms of E-ll-
tetradecenal, the major component of the spruce budworm
pheromone, for four hours at a flow rate of 70 liters per
hour. The air stream was then passed through a column of
Porapak Q (0.2 grams). The Porapak Q was extracted with one
ml of hexane for two minutes, the hexane evaporated (about 5
minutes) and the aldehyde dissolved in water. The total
recovery of pheromone as analysed by the bioluminescent assay
was 25~ using this method.
In other cases, the pheromonic aldehyde was recovered
by subjecting an atmospheric sample to a cold trap (dry ice
acetone, minus 70C) to condense the aldehyde and other
residual materials therein, followed by preparation of an
aqueous solution thereof. Such methods have given recoveries
of the order of 15%.
EXAMPLE 1
.
This example is given to demonstrate the general
procedures according to the invention, but does not involve
the use of hydroxylamine as an aldehyde scavenger.
The aqueous solution sample under test was injected
113~6~;~
into the assay mixture containing luciferase and reduced
flavine mononucleotides which have been reduced in situ with
sodium dithionite. This resulted in a rapid rise in
luminescence to a maximum, dependent upon the amount of
luciferase and pheromone aldehyde. Since excess mononucleotide
or oxygen is rapidly removed by chemical oxidation or reduction
respectively, the light intensity subsequently decays, dependent
primarily on the turnover rate of the enzymes intermediate
formed at the start of the reaction. Figure 1 illustrates
the bioluminescent response of Beneckea Harveyi luciferase
- to the isomers (E and Z) of the spruce budworm pheromone,
ll-tetradecenal, determined in the procedure generally
outlined above. The aldehyde (21 ng) in 1.0 ml of water at
22C was injected into 1.0 ml of 0.05 M of phosphate, 0.05 M
Of mercaptoethanol, pH 7.0, containing luciferase (about 10
micrograms) and S x 10 5M reduced flavine mononucleotide
(reduced with about 0.5 milligrams of sodium dithionite). The
light was detected with a photomultiplier tube and recorded
graphically. One unit of luminescence corresponds to 5.5 x 10 9
quanta per second based on the standard of Hastings and Weber
"J. OPT. SOC. AM., Volume 53, pages 1410-1415, (1963)." All
aldehyde stocks were prepared in dimethyl formamide and stored
at 4C. Luciferase was purified to homogeneity from the
bioluminescent bacterium Beneckea harveyi~ The solid line
represents the response of E-ll-tetradecenal and the dotted
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1134164Z
line represents the response of Z-ll-tetradecenal, of the
spruce budworm pheromone.
Table I below shows the results of conducting the
procedure described above on samples of the three listed
aldehydes. Each aldehyde was assayed to give a curve as
shown in Fig. l, and the maximum luminescence and the time
required for light emission to decay from 80~ to 40% of its
maximum of intensity recQrded. The values given in
Table I for each aldehyde are the average of between 10 and 14
determinations.
TABLE I
Bioluminescent Response of Luc;ferase to Spruce Bud~Jorm Pheromone
Maximum Half-time (sec) for
Aldehyde Luminescence (+ s.d.)Luminescent Decay (+ s.d.)
Tetradecanal 16 + 3 0.71 + .04
E-ll-Tetradecanal 16 + 2 0.79 + .09
Z~ Tetradecanal 13 + 1 0.99 + .07
.
EXAMPLE II
Two and three day old Eastern spruce budworms,
Choristoneura Fumiferana, maintained in continuous light,
were excised between 15 and 17 hours, EST, and extracted with
microliters of heptane for 10 minutes. The heptane extract
was directly transferred to a 20 ml glass vial, evporated under
113~642
low vacuum for 2 minutes to remove the heptane, vortexed for
10 seconds with 10 ml of water, and then assayed 10 minutes
later by the assay procedure according to the invention, i.e.
as described in Example 1 but with 0.01 M hydroxylamine
present in the assay mixture to lower the endogeneous activity.
The results are given in Figure 2 and Table II, In Figure 2,
the maximum luminescence is plotted versus the amount of
added E~ tetradecenal. Each point is the average of two
assays with the bars representing the range of the experimental
data. The abdomen samples in Table II refer to the tissue in
immediate proximity to the gland with the mass of material
analysed being approximately twice that for the gland. Each
budworm sample was analysed at least twice, the average value
corrected for background response for reference heptane samples
containing no budworm material and then converted into nanograms
based on the luminescent response of standards of E-ll-
tetradecenal in heptane carried through the identical process.
TABLE II
Pheromone Levels in the Spruce Budworm
. Average Amount
Number of Per Budworm
- Budworms Analyzed nq + s.d. Range (ng)
Female Gland 32 2.3 + 1.6 0.2 - 5.7
Female Abdomen 6 0.1 ~ 0.07 0.0 - 0.2
Female Head/Antennae 6 0.2 + 0.07 0.1 - 0.3
I Male Head/Antennae 6 0.1 + 0.06 0.0 - 0.2
113464Z
The results in ~igure 2 indicate that amounts of
pheromone as low as lO0 fentomoles can be measured according
to the process of the present in~vention, with the elimination
of the background aldehyde as described. Whilst these specific
experiments in Table II were conducted on extract from insect
budworms, it is clear that essentially similar results are
obtained on atmospherically collected samples, of the same
order of concentration.
The capability to detect very low amounts of phero-
mones using the luminescence assay according to the present
invention is of major advantage for studying the syntheses,
regulation and release of the pheromone from the female of the
species such as the spruce budworm. The assay is sensitive
enough quantitatively to measure the pheromone levels in the
gland of a single spruce budworm. The results show that the
amount of pheromone obtained from a heptane extract of the
gland of a single spruce budworm moth is in the range of 1-25
picamoles. In contrast, heptane extracts of the body of the
same insect contained little if any pheromone. The measurement
of pheromones by the bioluminescent assay process according to
the present invention, as applied to pheronome release into
the air, has the advantages of rapidity, sensitivity and ease of
quantitation. Trapping of the atmospheric pheromones by
1~3~6 ~Z
condensation with water in cold traps can be undertaken, since
the presence of water does not interfere wtih the analysis of
samples by this method and the pheromones are relatively
stable in water.
Whilst the invention has been described in reference
to certain specific examples and procedures, it will be
appreciated that it is not to be limited in scope to the
precise experimental conditions and procedure described.
The scope of the invention is limited only by the scope of
the appended claims.
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